Among several long-term objectives the following are chosen for the proposed grant period: (1) To purify and characterize Bam HI and Bgl I methylases from available endonuclease preparation side fractions. Pending on the purification and characterization of the two methylases parallel experiments are planned with the methylases. (2) To elucidate details of specificity changes for Bam HI activity upon changing the reaction environment. To determine DNA site at which cleavage occurs by Bam HI-2. To evaluate the effect of changes in reaction environment on the substate using biochemical and biophysical techniques. (3) To crystalize Bam HI and Bgl I (microcrystals for Bam HI have been obtained) in order to study their structure by X-ray diffraction. (4) To clone the structural genes for the Bam HI and Bgi I restriction endonucleases. (5) To determine inhibition patterns for Bam HI and Bgl I using subpalindromic sequences to differentiate between binding and catalytic sites. (6) To monitor conformation using intrinsic flouorescence of the endonucleases under conditions that will promote interaction with DNA. To determine the endonuclease/substrate ratio using fluorescent probes.